Inhibitor of effects of ultraviolet light on melanocytes

Abstract

The purpose of the present invention is to suppress the effect of ultraviolet light on melanocytes. Upon performance of gene expression analysis with a focus on the olfactory receptor OR2L13 expressed by melanocytes, it was discovered that addition of (−)-carvone, a ligand of OR2L13, increased the expression of genes related to suppression of active oxygen, suppression of the production of inflammatory cytokines or chemokines, suppression of senescence, and / or degradation of abnormal proteins. As a result of said discovery, the present invention provides an inhibitor of the effects of ultraviolet light on melanocytes that includes (−)-carvone.

Description

Inhibitor of the effects of ultraviolet light on melanocytes

[0001] The present invention relates to a technique for suppressing the effects of ultraviolet light on melanocytes. By suppressing the effects of ultraviolet light on melanocytes, the effects of aging such as pigmentation can be suppressed.

[0002] Melanocytes are activated by ultraviolet light and / or melanocyte-stimulating hormone (MSH) secreted by surrounding keratinocytes to produce melanin, which contributes to skin color, age spots, etc. Melanocytes are migratory cells and have been reported to express olfactory receptors. However, the function of olfactory receptors in melanocytes is still being elucidated.

[0003] Olfactory receptors are G protein-coupled proteins primarily expressed in olfactory cells. Humans have 396 functional olfactory receptor genes. While receptors typically function by binding to specific ligands, olfactory receptors bind to specific structures of odor molecules, allowing not just one molecule but multiple molecules to function as ligands for the olfactory receptor and activate it. This allows organisms to detect a variety of odors. Olfactory receptors are primarily expressed in olfactory cells, but they are not necessarily found exclusively in olfactory cells; cells other than olfactory cells are known to express olfactory receptors. For example, sperm express an olfactory receptor called MOR23 (mouse) or hOR17-4 (human), and these receptors are thought to be involved in sperm chemotaxis. Furthermore, an olfactory receptor called MOR23 expressed in mouse muscle cells has been reported to be involved in muscle fiber repair (Non-Patent Document 1: Dev Cell., 17, 649-661, (2009)). Kidney cells have been reported to express multiple olfactory receptors and to respond to a variety of stimuli (Non-Patent Document 2: Proc Natl Acad Sci USA., 10, 2059-2064, (2009); Non-Patent Document 3: Proc Natl Acad Sci USA., 110, 4410-4415, (2013)). In addition, it has been reported that a receptor called OR2AT4 expressed by keratinocytes in the skin is involved in wound healing (Non-Patent Document 4: J Invest Dermatol., 134, 2823-2832, (2014)).

[0004] Dev Cell., 17, 649-661, (2009)Proc Natl Acad Sci USA., 10, 2059-2064, (2009)Proc Natl Acad Sci USA., 110, 4410-4415, (2013)J Invest Dermatol., 134, 2823-2832, (2014)

[0005] The purpose is to suppress the effects of ultraviolet rays on melanocytes.

[0006] Focusing on the olfactory receptor OR2L13 expressed by melanocytes, the present inventors added (-)-carvone, a ligand for OR2L13, and performed gene expression analysis. They found that the expression of genes involved in the suppression of reactive oxygen species, the suppression of the production of inflammatory cytokines or chemokines, the suppression of aging, and / or the degradation of abnormal proteins was increased, leading to the present invention. Therefore, the present invention relates to the following:

[0007] [1-1] An agent for suppressing the effects of ultraviolet light on melanocytes, comprising (-)-carvone. [1-2] A method for suppressing the effects of ultraviolet light on melanocytes, comprising administering (-)-carvone to a subject in whom suppression of the effects of ultraviolet light is desired. [1-3] (-)-carvone for use in treating ultraviolet light-induced diseases through suppression of the effects of ultraviolet light on melanocytes. [1-4] Use of (-)-carvone for producing an agent for suppressing the effects of ultraviolet light on melanocytes. [2] The invention described in any one of items 1-1 to 1-4, wherein the effects of ultraviolet light are at least one selected from the group consisting of production of reactive oxygen species, inflammation, aging, and accumulation of abnormal proteins. [3] The invention described in item 2, wherein the effects of ultraviolet light due to production of reactive oxygen species are suppressed by increasing oxidative stress response-related genes. [4] The invention described in item 3, wherein the oxidative stress response-related gene is at least one selected from the group consisting of SOD1 and GSR. [5] The invention according to Item 4, wherein the amount of NADH is increased or the amount of succinic acid is decreased. [6] The invention according to Item 2, wherein the effect of ultraviolet light on inflammation is suppressed by reducing the expression of inflammatory cytokines or chemokines. [7] The invention according to Item 6, wherein the inflammatory cytokine or chemokine is at least one selected from the group consisting of IL-16, CCL2, and CXCL1. [8] The invention according to Item 2, wherein the effect of ultraviolet light on aging is suppressed by reducing the gene expression of mTOR and TGFβ. [9] The invention according to Item 8, wherein the production of adenosine and guanosine is promoted.

[10] The invention according to any one of Items 1 to 9, wherein (-)-carvone acts via the olfactory receptor OR2L13.

[11] The invention according to Item 2, wherein the inhibitor reduces abnormal proteins by increasing the expression of proteasome-related genes.

[12] The invention according to Item 11, wherein the proteasome-related gene is at least one selected from the group consisting of PSMA5, PSMD4, PSMB, PSMC, PSMD, ADRM1, and POMP.

[13] A skin whitening agent comprising the inhibitor according to Item [1-1]. [14-1] An antioxidant power enhancer comprising (-)-carvone.[14-2] A method for improving antioxidant capacity, comprising administering (-)-carvone to a subject in whom improvement of antioxidant capacity is desired. [14-3] (-)-carvone for use in treating oxidative stress-induced diseases through improvement of antioxidant capacity. [14-4] Use of (-)-carvone for producing an agent for improving antioxidant capacity. [15-1] An agent for promoting expression of oxidative stress response-related genes, comprising (-)-carvone. [15-2] A method for improving expression of oxidative stress response-related genes, comprising administering (-)-carvone to a subject in whom improvement of antioxidant capacity is desired. [15-3] (-)-carvone for use in treating oxidative stress-induced diseases through promotion of expression of oxidative stress response-related genes. [15-4] Use of (-)-carvone for producing an agent for improving oxidative stress response-related genes. [16-1] An anti-inflammatory agent comprising (-)-carvone. [16-2] A method for suppressing inflammation in a subject in whom suppression of inflammation is desired, comprising administering (-)-carvone. [16-3] (-)-carvone for use in the treatment of inflammatory diseases. [16-4] Use of (-)-carvone for the production of an anti-inflammatory agent. [17-1] An inhibitor of inflammatory cytokine or chemokine expression, comprising (-)-carvone. [17-2] A method for suppressing inflammatory cytokine or chemokine expression in a subject in whom suppression of inflammation is desired, comprising administering (-)-carvone. [17-3] (-)-carvone for use in the treatment of inflammatory diseases via the suppression of inflammatory cytokine or chemokine expression. [17-4] Use of (-)-carvone for the production of an anti-inflammatory agent.

[18] The invention according to items 15-1 to 15-4, wherein the inflammatory cytokine or chemokine is at least one selected from the group consisting of IL-16, CCL2, and CXCL1. [19-1] An inhibitor of aging-associated gene expression, comprising (-)-carvone. [19-2] A method for inhibiting aging-associated gene expression in a subject in whom inhibition of aging is desired, comprising administering (-)-carvone. [19-3] (-)-carvone for use in treating aging-associated diseases via inhibition of aging-associated gene expression. [19-4] Use of (-)-carvone for producing an inhibitor of aging-associated gene expression.

[20] The invention according to Items 17-1 to 17-4, wherein the aging-related gene is mTOR or TGFβ. [21-1] An agent for promoting the production of adenosine and / or guanosine, comprising (-)-carvone. [21-2] A method for promoting the production of adenosine and / or guanosine, comprising administering (-)-carvone to a subject in whom promotion of adenosine and guanosine production is desired. [21-3] (-)-carvone for use in the treatment of aging-related diseases via promotion of adenosine and / or guanosine production. [22-4] Use of (-)-carvone for the manufacture of an agent for promoting the production of adenosine and / or guanosine. [23-1] An agent for promoting the expression of proteasome-related genes, comprising (-)-carvone. [23-2] A method for promoting the expression of proteasome-related genes, comprising administering (-)-carvone to a subject in whom promotion of proteasome-related gene expression is desired. [23-3] (-)-Carvone for use in treating diseases associated with abnormal protein accumulation via promoting expression of proteasome-related genes. [23-4] Use of (-)-carvone for producing an agent for promoting expression of proteasome-related genes.

[24] The invention according to Items 23-1 to 23-4, wherein the proteasome-related gene is at least one selected from the group consisting of PSMA5, PSMD4, PSMB, PSMC, PSMD, ADRM1, and POMP.

[0008] By applying (-)-carvone, the effects of ultraviolet rays on melanocytes can be suppressed.

[0009] Figure 1 shows the expression of the olfactory receptor OR2L13 in melanocytes (A: PCR, B: immunofluorescence staining photograph). Figure 2 shows the classification of genes whose expression increased when (-)-carvone was added to melanocytes, as detected by RNA-seq for all genes. Genes involved in antioxidant responses are underlined in gray, and genes contributing to anti-inflammatory responses are underlined in black. Figure 3 shows graphs showing the changes in expression of specific genes (SOD1 (A), GSR (B)) in cells when (-)-carvone was added. Figure 4 shows the changes in the production of two components that showed large fluctuations in the results of metabolome analysis of cells when (-)-carvone was added (NADH (A) and succinic acid (B)). Figure 5 shows the changes in expression of specific genes (mTOR (A) and TGFβ (B)) in cells when (-)-carvone was added. Figure 6 shows the changes in the production amounts of two components that showed large fluctuations in the results of metabolome analysis of cells when (-)-carvone was added (adenosine (A) and guanosine (B)). Figure 7 shows the changes in expression of specific genes (IL-16 (A), CCL2 (B), and CXCL1 (C)) in cells when (-)-carvone was added. Figure 8 shows the changes in expression of specific genes (IPSMA5 (A) and PSMD4 (B)) in cells when (-)-carvone was added.

[0010] The present invention relates to an agent for suppressing the effects of ultraviolet light on melanocytes, which contains (-)-carvone. By suppressing the effects of ultraviolet light, melanin production in melanocytes is inhibited, thereby achieving effects such as whitening and prevention of spots and dullness.

[0011] (-)-Carvone has the following formula: (-)-Carvone is a volatile compound having the formula (I). It is an aromatic compound found in caraway and spearmint. (-)-Carvone is a specific ligand for the olfactory receptor OR2L13. Therefore, preparations containing (-)-carvone may contain (-)-carvone directly, or may contain an extract of a plant containing (-)-carvone, such as caraway or spearmint.

[0012] When ultraviolet rays are irradiated onto the skin, they cause inflammation, redness, swelling, blisters, peeling, and sunburn, resulting in darkening. In the long term, sunburn can lead to skin aging, and in particular, the degeneration of the dermis layer and pigmentation of the epidermis layer caused by ultraviolet rays is called photoaging. UV rays act directly on DNA and proteins, and also act indirectly by generating reactive oxygen species. These effects of UV rays interact with each other to contribute to sunburn and aging.

[0013] UV rays directly affect the genomic DNA of cells, particularly skin cells, causing DNA damage. Damage to genomic DNA can occur through the formation of dimers between adjacent bases or through breakage. Damage to genomic DNA is repaired by the cell's own repair mechanism, but if some of the damage is repaired incorrectly and accumulates, cell activity declines, leading to aging.

[0014] Ultraviolet rays also directly affect proteins. Ultraviolet rays affect hydrogen bonds and other structures, and also cause structural changes in amino acid side chains, denaturing proteins. A wide variety of proteins are susceptible to denaturation by ultraviolet rays, but amino acids containing side chains highly reactive to ultraviolet rays, such as collagen rich in proline and histidine, are easily denatured by ultraviolet rays. While not intending to be limited by theory, amino acids highly reactive to ultraviolet rays often have absorption bands in the ultraviolet region, and examples include proline, histidine, tryptophan, tyrosine, and phenylalanine.

[0015] Ultraviolet rays generate reactive oxygen species both inside and outside cells. The generated reactive oxygen species denature proteins inside and outside the cells, increasing the amount of abnormal proteins and causing aging. Furthermore, reactive oxygen species generated near genomic DNA cause DNA breaks through the oxidation of bases, resulting in DNA damage.

[0016] In response to UV rays' direct action on DNA, direct action on proteins, and generation of reactive oxygen species, biological tissues and / or cells exhibit various physiological responses, such as cytokine secretion, activation of matrix proteinases, activation of melanocytes, and mechanisms for suppressing reactive oxygen species, ultimately inducing short-term responses such as inflammation and skin darkening, and long-term responses such as aging.

[0017] In the present invention, the effects of UV rays on melanocytes include, without intending to be limited by theory, the generation of reactive oxygen species, inflammation, and aging caused by UV rays. Reactive oxygen species act directly on melanocytes, causing accumulated damage. Furthermore, melanocytes exposed to UV rays secrete inflammatory cytokines and / or chemokines, causing inflammation in the surrounding area. Melanocytes are activated by melanocyte activating factors secreted by surrounding skin cells due to the action of UV rays. This induces melanin production and the expression of aging factors, leading to aging phenomena such as pigmentation.

[0018] More specifically, an inhibitor of the effects of UV rays on melanocytes containing (-)-carvone can upregulate proteins involved in the elimination of reactive oxygen species. Examples of such proteins include superoxide dismutase 1 (SOD1) and glutathione disulfide reductase (GSR). Without intending to be limited by theory, an inhibitor of the effects of UV rays on melanocytes containing (-)-carvone reduces oxidative stress in melanocytes by promoting the expression of oxidative stress response-related genes, such as superoxide dismutase 1 (SOD1) and glutathione disulfide reductase (GSR). This increases the amount of NADH in the cells and reduces the amount of succinic acid in the cells related to oxidative stress. Therefore, an inhibitor of the effects of UV rays on melanocytes can be referred to as an oxidative stress response-related gene expression promoter or an antioxidant potency enhancer. This can suppress reactive oxygen species and physiological effects triggered by reactive oxygen species. Physiological effects triggered by reactive oxygen include the production of abnormal proteins, cytokine production, and DNA damage, and these physiological effects can be suppressed.

[0019] SOD1 is an enzyme that breaks down reactive oxygen species generated within cells. SOD1 has a metal ion in its active center and is mainly localized in the cytoplasm. SOD1 breaks down superoxide anions (.O2 - ) with H cations to convert them into oxygen and hydrogen peroxide, thereby reducing oxidative stress.

[0020] GSR is one of the glutathione metabolic enzymes. GSR is involved in the following reaction: By promoting the expression of GSR, the antioxidant glutathione can be regenerated, thereby reducing oxidative stress.

[0021] NADH is nicotinamide adenine dinucleotide. It accepts hydrogen atoms and electrons, and oxidized NAD+ is converted to reduced NADH by accepting one hydrogen ion and two electrons. Therefore, the amount of reduced NADH present in cells is an indicator of oxidative stress, and the greater the amount of NADH present, the less exposed the cells are to oxidative stress.

[0022] Succinic acid is one of the compounds that make up the TCA cycle. It has been reported that succinic acid levels are elevated in patients with sepsis, and that succinic acid contributes to inflammatory responses, hypoxia, and the production of reactive oxygen species (International Immunopharmacology vol. 110, 2022, 109065). Since increased intracellular succinic acid promotes the production of reactive oxygen species, a low level of succinic acid in cells is an indicator of oxidative stress, and the lower the amount of succinic acid present, the less exposed the cells are to oxidative stress. Succinic acid may exist in the form of a salt, particularly a sodium or potassium salt, depending on the intracellular pH.

[0023] Because the action of (-)-carvone increases the amount of NADH and decreases the amount of succinic acid in melanocytes, (-)-carvone can reduce oxidative stress. Therefore, another aspect of the present invention relates to an oxidative stress-reducing agent containing (-)-carvone, which can act particularly on melanocytes.

[0024] In another aspect, an agent for suppressing the effects of ultraviolet light on melanocytes, comprising (-)-carvone, can more specifically reduce the expression of inflammatory cytokines or chemokines in melanocytes. Examples of inflammatory cytokines or chemokines include, but are not limited to, IL-16, CCL2, CXCL1, IL-1β, IL-5, IL-11, IL-25, and CXCL6. (-)-Carvone suppresses the expression of inflammatory cytokines or chemokines in melanocytes. Therefore, another aspect of the present invention may relate to an inflammation suppressor comprising (-)-carvone. In particular, it can suppress inflammation involving melanocytes, and can treat, improve, or prevent, for example, skin inflammation, sunburn, atopic dermatitis, and the like. Furthermore, since skin inflammation is also involved in the production and deposition of melanin pigment, suppressing the expression of inflammatory cytokines or chemokines can also exert a whitening effect. In addition, (-)-carvone can prevent or treat inflammatory diseases induced by inflammatory cytokines or chemokines.

[0025] Interleukin-16 (IL-16) is a cytokine that was originally reported as a factor that attracts activated T cells and is also called lymphocyte chemotactic factor (LCF). It is expressed as pro-IL-16 and is released extracellularly as a mature interleukin following the action of caspases and other enzymes. The released IL-16 attracts CD4+ cells such as monocytes, eosinophils, and dendritic cells, contributing to the initiation of inflammation.

[0026] CCL2 is a chemokine belonging to the CC chemokine family and is also known as MCP-1 or SCYA2. CCL2 is not only anchored to the cell membrane but is also secreted by monocytes, macrophages, and dendritic cells. It has the ability to attract monocytes and basophils, contributing to the initiation of inflammation. Furthermore, CCL2 has been suggested to be involved in inflammatory diseases involving monocyte infiltration, such as psoriasis, rheumatoid arthritis, and atherosclerosis.

[0027] CXCL1 is a chemokine belonging to the CXC chemokine family and a ligand for chemokine receptor 2 (CXCR2). CXCL1 acts as a chemoattractant that attracts immune cells such as neutrophils and non-hematopoietic cells to sites of injury or infection, thereby contributing to the regulation of inflammatory responses.

[0028] The agent for suppressing the effects of ultraviolet light on melanocytes, which contains (-)-carvone of the present invention, can reduce the expression of aging-related genes. Examples of aging-related genes include mTOR and TGFβ. This can suppress the effects of ultraviolet light on aging. By suppressing the expression of aging-related factors, an anti-aging effect is brought about in melanocytes, and melanocyte activity is optimized.

[0029] mTOR is a kinase that has been identified as a molecular target of rapamycin. mTOR acts on various signaling-related factors, such as eIF4G, PPAR, and HIF-1, and contributes to the control of various physiological functions. These physiological functions are diverse, including cell proliferation, autophagy, proliferation, and lipid metabolism. Recent reports have shown that mTOR inhibits autophagy and promotes protein synthesis. However, this condition can be considered a state of high DNA damage and cellular stress, causing the accumulation of cellular damage and contributing to aging (Int. J. Mol. Sci. (2019), 20(11), 2774, Nature (2013) vol. 493, pp. 338-345). Therefore, it has been suggested that suppressing mTOR expression can halt the progression of aging and increase survival.

[0030] Transforming growth factor β (TGFβ) is a secreted protein that forms a TGFβ complex and binds to the TGFβ receptor. The TGFβ receptor is a receptor-type serine / threonine kinase, and upon binding with TGFβ, it activates a signal transduction cascade, resulting in various physiological effects such as differentiation, chemotaxis, proliferation, and immune activation. It is known to be one of the factors that contribute to skin aging.

[0031] Adenosine and guanosine are basic compounds formed by the binding of adenine and guanine, respectively, to ribose, and are phosphorylated to form nucleic acids. In addition to being building blocks of nucleic acids, adenosine and guanosine themselves are known to have physiological effects, including the ability to inhibit cellular aging.

[0032] The proteasome is composed of the α subunit PSMA, the β subunit PSMB, the ATPase subunit PSMC, and the non-ATPase subunit PSMD, which combine to form the 20S proteasome, 19S proteasome, and 11S proteasome. These proteasomes further combine to form enzyme complexes involved in protein degradation via the ubiquitin-proteasome system. The subunits PSMA to PSMD have multiple subtypes, which can be referred to as proteasome-related genes. Examples of proteasome-related genes include, but are not limited to, PSMB, PSMC, PSMD, ADRM1, POMP, and their subtypes. PSMA4 and PSMD5 may be used as examples. Ubiquitin-tagged proteins are directed to the proteasome, where they undergo ubiquitin-dependent degradation. Protein ubiquitination can occur in various ways, including the ubiquitination of abnormally folded proteins, which are then degraded by the proteasome. Therefore, promoters of proteasome-related gene expression can improve or mitigate the accumulation of abnormal proteins by promoting the degradation of abnormal proteins. This has the effect of improving cellular aging. Abnormal proteins may be generated by the direct action of ultraviolet light or reactive oxygen species, or by other causes.

[0033] The agent for suppressing the effects of ultraviolet light on melanocytes according to the present invention can suppress at least one phenomenon selected from the group consisting of the generation of reactive oxygen species, inflammation, and aging, which are the effects of ultraviolet light. The agent for suppressing the effects of ultraviolet light, the whitening agent, the antioxidant power enhancer, the agent for promoting the expression of oxidative stress response-related genes, the anti-inflammatory agent, the agent for suppressing the expression of inflammatory cytokines or chemokines, the agent for suppressing the expression of inflammatory cytokines or chemokines, the agent for suppressing the expression of aging-related genes, and the agent for promoting the production of adenosine and guanosine according to the present invention can each be used for health maintenance, cosmetic purposes, or medical purposes.

[0034] The concentration and dosage form of the agent of the present invention can be selected as desired to achieve the desired effects, i.e., UV protection, whitening, improved antioxidant activity, oxidative stress reduction, anti-inflammation, anti-aging, etc. (-)-Carvone can be incorporated into foods, cosmetics, pharmaceuticals, or quasi-drugs. When incorporated into foods, cosmetics, quasi-drugs, or pharmaceuticals, the corresponding action, use, and / or efficacy can be labeled. In another embodiment, medical or therapeutic use can be excluded. In such cases, it is particularly preferable that it is related to beauty treatments. When incorporated into foods, it may be incorporated into dietary supplements such as supplements and energy drinks, or functional food products. When incorporated into cosmetics, it may be incorporated into face or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, and gels, makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into pharmaceuticals, it may be administered orally or parenterally, for example, transdermally. When administered transdermally, it may be formulated into a topical skin preparation. The effects of ultraviolet light can be suppressed by using foods, cosmetics, pharmaceuticals, and quasi-drugs containing (-)-carvone. Specifically, the production of active oxygen, inflammation, and / or aging can be suppressed, thereby improving wrinkles, age spots, and / or sagging, or exhibiting a whitening effect. The foods, cosmetics, or pharmaceuticals containing (-)-carvone of the present invention can be administered over a long period of time. From the viewpoint of achieving an anti-aging effect, they may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. The upper limit is not particularly limited, but may be several years or less, for example, one year or less.

[0035] (-)-Carvone may be administered to, for example, a subject in need of suppression of the effects of ultraviolet rays. Such a subject may be a subject in need of suppression of at least one phenomenon selected from the group consisting of production of reactive oxygen species, inflammation, and aging. Furthermore, (-)-Carvone may be administered to a subject suffering from skin conditions such as wrinkles, age spots, and sagging skin, or skin aging, or a subject suffering from excessive inflammation or oxidative stress.

[0036] Taking into consideration its efficacy and safety, (-)-carvone can be used in different concentration ranges, and such concentrations can be determined by conducting efficacy tests and / or safety tests. As an example, from the perspective of incorporation into cosmetics and quasi-drugs, (-)-carvone can be incorporated at 0.08% to 2.5%. In particular, from the perspective of incorporation into cosmetics, pharmaceuticals, and quasi-drugs for topical use on the skin, (-)-carvone can be incorporated at preferably 0.008% or more, and more preferably 0.01% or more. From the perspective of incorporation into fragrances and miscellaneous goods, (-)-carvone can be incorporated at preferably 2.5% or less, and more preferably 1.2% or less. When incorporated into foods or pharmaceuticals, different concentration ranges may be used.

[0037] The topical skin preparation is not particularly limited as long as it is applicable to the skin, and any dosage form can be used, for example, a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a topical skin preparation, bases and excipients typically used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0038] All documents mentioned herein are incorporated by reference in their entirety.

[0039] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0040] Example 1: Expression of OR2L13 gene in melanocytes Melanocytes (Kurabo) were cultured at 5.0 × 10 4Melanocytes were seeded at 1000 cells / mL onto 6-well plates and cultured in DermaLife BM medium (Kurabo Industries, Ltd.) at 37°C under a 5% CO2 atmosphere for 48 hours. After incubation, cells were harvested using RLT buffer (Qiagen), and RNA was purified from the harvested cells using an RNeasy Mini Kit (Qiagen). The purified RNA was reverse transcribed using SuperScript IV VILO Master Mix (Thermo Fisher) and amplified using OR2L13 expression primers. The results are shown in Figure 1(A). Cultured melanocytes were fixed with 4% PFA and incubated with a 200-fold diluted anti-OR2L13 antibody (Invitrogen) and DAPI as the primary antibody. Fluorescent staining was performed using a fluorescently labeled anti-rabbit antibody as the secondary antibody. The stained samples were observed under a fluorescence microscope (Figure 1(B)).

[0041] Example 2: Transcriptome analysis of melanocytes Melanocytes (Kurabo) were used at 5.0 × 10 4Cells were seeded at 1000 μM per well onto a 6-well plate and cultured in DermaLife BM medium (Kurabo Industries, Ltd.) at 37°C under a 5% CO2 atmosphere for 48 hours. (-)-Carvone (Tokyo Chemical Industry Co., Ltd.) was added at a concentration of 1000 μM, and the cells were cultured in DermaLife BM medium at 37°C under a 5% CO2 atmosphere for 18 hours. After culture, the cells were harvested using RLT buffer (Qiagen), and RNA was purified from the harvested cells using an RNeasy Mini Kit (Qiagen). The purified RNA was sequenced using Novaseq (Illumina), and the sequence data was used to calculate the expression levels of each transcript. The resulting gene list was used to extract differentially expressed genes and perform pathway analysis using iDEP.96 (South Dakota State University). The results are shown in Figure 2. As genes whose expression levels were increased in melanocytes by the application of (-)-carvone, we focused on genes involved in cellular responses to chemical stress (R-HSA-9711123), genes involved in the NRF2 pathway (WP2884), genes involved in photodynamic therapy-induced NFE2L2 (NRF2) survival signaling (WP3612), genes involved in oxidative stress response (GO:0006979), genes involved in responses to toxic substances (GO:0009636), and genes involved in glutathione metabolic processes (GO:0006749). Among these, the genes involved in the NRF2 pathway (WP2884), the genes involved in photodynamic therapy-induced NFE2L2 (NRF2) survival signaling (WP3612), the genes involved in oxidative stress response (GO:0006979), and the genes involved in glutathione metabolic processes (GO:0006749) can be considered to contribute to anti-inflammatory responses. Additionally, the genes involved in cellular responses to chemical stress (R-HSA-9711123) and the genes involved in responses to toxic substances (GO:0009636) can be considered to contribute to anti-inflammatory responses.As genes extracted as those whose expression levels were changed, attention was focused on SOD1 and GSR (FIG. 3), mTOR and TGFβ (FIG. 5), as well as IL-16, CCL2, and CXCL1 (FIG. 7).

[0042] Example 3: Metabolome analysis in melanocytes Melanocytes (Kurabo) were used at 5.0 × 10 4 The cells were seeded at 1000 μM in a 6-well plate and cultured in DermaLife BM medium (Kurabo Industries, Ltd.) at 37°C in a 5% CO2 atmosphere for 48 hours. (-)-Carvone (Tokyo Chemical Industry Co., Ltd.) was added at a concentration of 1000 μM, and the cells were cultured in DermaLife BM medium at 37°C in a 5% CO2 atmosphere for 18 hours. After culture, the cells were washed with PBS and then the cell suspension was recovered using methanol. The recovered cell suspension was subjected to liquid-liquid partitioning to fractionate hydrophilic and hydrophobic components. The fractionated hydrophilic components were concentrated to dryness, redissolved, and subjected to an analyzer. The analytical method used was a hydrophilic interaction and anion exchange mixed polymer column (unified-HILIC / AEX, RESONAC) with an LC-MS / MS analysis system consisting of a liquid chromatograph and a triple quadrupole mass spectrometer (LCMS8060, Shimadzu Corporation). Multiple reaction monitoring (MRM) of 400 hydrophilic metabolites and targeted metabolome analysis were performed. Gene-Metabolite Interaction Network analysis was performed using MetaboAnalyst to analyze the experimental results. We focused on NADH and succinic acid (Figure 4), adenosine, and guanosine, which showed metabolic changes in melanocytes due to the action of (-)-carvone (Figure 6).

Claims

1. Inhibitors of the effects of ultraviolet light on melanocytes, including (-)-carvone.

2. The inhibitor according to claim 1, wherein the effect of ultraviolet rays is at least one selected from the group consisting of the production of reactive oxygen species, inflammation, aging, and accumulation of abnormal proteins.

3. The inhibitor according to claim 2, which inhibits the effects of ultraviolet light due to the production of reactive oxygen species by increasing the expression of oxidative stress response-related genes.

4. The inhibitor according to claim 3, wherein the oxidative stress response-related gene is at least one selected from the group consisting of SOD1 and GSR.

5. The inhibitor of claim 4, wherein the inhibitor increases the amount of NADH or decreases the amount of succinic acid.

6. The inhibitor of claim 2, wherein the inhibitor inhibits the effects of ultraviolet light on inflammation by reducing the expression of inflammatory cytokines or chemokines.

7. The inhibitor according to claim 6, wherein the inflammatory cytokine or chemokine is at least one selected from the group consisting of IL-16, CCL2, and CXCL1.

8. The inhibitor according to claim 2, which inhibits the effects of ultraviolet light on aging by reducing gene expression of mTOR and TGFβ.

9. The inhibitor of claim 8, wherein the inhibitor promotes the production of adenosine and guanosine.

10. The inhibitor according to any one of claims 1 to 9, wherein (-)-carvone acts via the olfactory receptor OR2L13.

11. The inhibitor according to claim 2, which reduces abnormal proteins by increasing the expression of proteasome-related genes.

12. The inhibitor according to claim 11, wherein the proteasome-related gene is at least one selected from the group consisting of PSMA5, PSMD4, PSMB, PSMC, PSMD, ADRM1, and POMP.

13. A skin whitening agent comprising the inhibitor according to any one of claims 1 to 12.

14. (-)-Antioxidant enhancer containing carvone.

15. (-)-Carvone is a promoter of oxidative stress response-related genes.

16. The expression promoter of claim 13, wherein the oxidative stress response-related gene is SOD1 or GSR.

17. (-)-Anti-inflammatory agents, including carvone.

18. Inhibitors of inflammatory cytokine or chemokine expression, including (-)-carvone.

19. The inhibitor of inflammatory cytokine or chemokine expression according to claim 18, wherein the inflammatory cytokine or chemokine is at least one selected from the group consisting of IL-16, CCL2, and CXCL1.

20. Inhibitors of aging-related gene expression, including (-)-carvone.

21. The inhibitor according to claim 20, wherein the aging-associated gene is mTOR or TGFβ.

22. An adenosine and / or guanosine production promoter, including (-)-carvone.

23. A promoter of proteasome-related gene expression, including (-)-carvone.

24. The proteasome-related gene expression promoter according to claim 23, wherein the proteasome-related gene is at least one selected from the group consisting of PSMA5, PSMD4, PSMB, PSMC, PSMD, ADRM1, and POMP.

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